Safety and Protocols

iGEM LAB PROCEDURES

Taq Polymerase Chain Reaction

Polymerase Chain Reaction Master Mix

Reagent	1x Mix (µL)	5x Mix (µL)
H2O	28	140
10x Buffer	5	25
2 mM dNTP	5	25
Forward Primer (2 mM)	5	25
Reverse Primer (2 mM)	5	25
50 mM MgCl2	1.5	7.5
Taq Polymerase	0.5	2.5
Total	50	250

NOTE: Taq is used because it is functional at relatively high temperatures. MgCl₂ is a salt that allows Taq Polymerase to function. Water is added in two portions (half added before all other reagents and half added after all other reagents) Please note that quantity of water decreases in proportion to the DNA template added so the total remains the same.

Thermocycler PCR Program

Temperature (°C)	Time (mins)
95.0	5:00
95.0	1:00
55.0*	0:30
72.0	1:00
72.0	10:00
4.0	∞

NOTE: * indicates that the temperature of the step is primer specific. Steps 2 through 4 are repeated 30 times. Step 4 is done at a rate of 1 min/1000 bp

Transformable Competent Cell Induction Protocol
This procedure was done using Top10 Competent cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.

1. Innoculate 5-10 mL LB at 37°C while shaking
2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37° while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours)
3. Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes
4. Resuspend pellet in 12.5 mL of cold CaCl₂ (50 mM) and leave on ice for 10 minutes
5. Centrifuge at 10 000 rpm at 4°C for 2 minutes and resuspend in 2 mL of cold CaCl₂ (50 mM, 15% glycerol solution)
6. Leave on ice for at least 30 minutes and then aliquot 200 uL and freeze at -80°C

Bacterial Transformation Protocol

1. Thaw Competent Cells
2. Add 10-100 ng of DNA
3. Ice solution for 30 minutes
4. Heat shock solution (5 minutes at 37°C or 2 minutes at 42°C)
5. Ice solution for 5 minutes
6. Recover with 250 µL of SOC (30 minutes for Ampicilin resistant plasmids and 60 minutes for kanamycin resistant plasmids)
7. Centrifuge for 5 seconds at 14 000 rpm and concentrate solution to 100 µL
8. Plate 20-50 µL onto each spread plate

Restriction Digest
This protocol is part of the Construction Protocol. Start by selecting one of the parts you wish to combine as the vector (the plasmid will be kept) and the other part as the insert. The two parts will need to be mixed separately at the beginning. The parts must be kept separate for the digestion period.

Add to the Insert Tube:

• 600 ng of DNA (Calculate this from the concentration of plasmid)
• 3.5 µL of 10x Buffer
• 0.5 µL of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)
• H₂O such that the volume of water and DNA in the tube is 30.5 µL and the total volume in each tube is 35 µL

Add to the Vector Tube:

• 250 ng of DNA (Calculate this from the concentration of plasmid)
• 3.5 µL of appropriate 10x Buffer
• 0.5 µL of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)
• HH₂O such that the volume of water and DNA in the tube is 30.5 µL and the total volume in each tube is 35 µL

1. Mix two tubes as indicated above
2. Put the tubes into a 37°C water bath for one hour
3. Place the tubes into an 80°C heating block for 20 minutes to heat-kill the enzymes in the tube
4. Freeze the parts until they are needed

Ligation
This protocol is part of the Construction Protocol. The tubes from the Restriction Digest should be removed from the freezer and thawed on ice before beginning ligation.

1. Mix 5 µL of the insert and 5 &microl: of the vector in a new tube
2. Clearly label the tubes as unligated, write the date and freeze the tubes in -20°C in case the transformation does not work
3. Add 10 µL of 2x Quick Ligase Buffer and 1 µL of Quick Ligase to the tube containing the mixed Insert and Vector
4. Let the tube sit at room temperature for 5 minutes
5. Transform this mix (all 21 µL) into Top10 Competent Cells

Miniprep Plasmid Preparation (GenElute)
This protocol is taken from the Sigma Aldrich distributed GenElute Miniprep Plasmid Preparation Kits. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer. We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice. Another company's kit was used occasionally to prep the plasmids but the two kits were never mixed.

1. Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol)
2. After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4°C
3. Discard the supernatant, while being careful not to discard any of the pellet
4. Resuspend the pellet in 200 µL of Resuspension Solution (with RNase A added) which is provided from the kit
5. Transfer the solution from a Falcon tube to a 1.5 µL microcentrifuge tube
6. Add 200 µL of Lysis Solution and invert gently to mix. Allow the mixture to clear for less than 5 minutes
7. Add 350 µL of Neutralization Solution and invert the tube 4-6 times to mix
8. Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The resulting solution will be known as the lysate
9. Add 500 µL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube
10. Transfer the lysate into the binding column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid
11. Centrrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube
12. Add 750 µL of Wash Solution with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube
13. Repeat Step 12 a second time with the same quantity of Wash Solution
14. Centrifuge the tube for 1 minute at 14 000 rpm to dry the column
15. Transfer the column to a new 1.5 µL microcentrifuge tube
16. Add 50 µL of double distilled water to the column and spin for 1 minute at 14 000 rpm
17. Use a spectrophotometer to measure the concentration and the purity of your plasmid

Agarose Gel Electrophoresis
This procedure is involved in tandem with a PCR. The first step involves the making of an agarose gel as indicated by the Making of Agarose Gel protocol.

1. Create an agarose gel tray
2. Place the gel tray into a gel dock where it will be run. Add TAE buffer such that the entire gel is covered
3. Make mixed tubes with 3 µL DNA, 2 µL Loading dye, and 15 µL water
4. Insert 10 µL of the mixture into each well with 5 µL of the 1KB ladder in the first hole
5. Place thecovering on top and set it to run at 90V
6. When the bands are approximately halfway through the gel, which should be around 35-40 minutes, turn off the electricity and remove the gel
7. Use a computer imager to take a picture of the gel

Making of Agarose Gel
The agarose gel is made in order to do agarose gel electrophoresis. The procedure is modified by the quantity of agarose added to the solution. The procedure here will detail the making of a 1.5% gel.

1. Measure out 1.5g of agarose
2. Add the agarose to 100 mL of TAE buffer. COver the beaker with saran wrap with a hole punched in it
3. Microwave the solution for 30 seconds and then swirl. Then, microwave for 1 minute at high power and swirl. Finally, microwave for 1 minute further and swirl
4. Take this solution to the fume hood and add 3 µL of ethidium bromide. Ethidium bromide is a suspected carcinogen so handle with care
5. Swirl the solution to allow the ethidium bromide to mix
6. Pour the solution into the tray. Use a 10 µL pipette tip to pop any bubbles that may result and insert the comb
7. Allow the solution to solidify and remove the comb

Rehydration of Registry DNA
The open source Parts Registry is where all parts are sent by iGEM teams each year. The distribution is done in three 384-well plates in dry DNA form. The DNA must be rehydrated, transformed into Top10 Competent cells and then plasmid prepped using the Miniprep Plasmid Prep Protocol as listed above before they are in usable DNA form.

1. Use a 10 µL pipette tip to puncture the aluminium foil covering of the desired well of DNA
2. After 5 minutes to allow the DNA to thaw, add 10 µL of double distilled water to the well and pipette up and down 3-4 times until the liquid comes up red
3. Take 2 µL of DNA and transform these into Top10 Competent Cells using the Transformation Protocol

Construction Technique
This protocol combines the Restriction Digest with the Ligation protocol as well as adding Antarctic Phosphatase Protocol.

Restriction Digest

Add to the Insert Tube:

• 600 ng of DNA (Calculate this from the concentration of plasmid)
• 3.5 µL of 10x Buffer
• 0.5 µL of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)
• H₂O such that the volume of water and DNA in the tube is 30.5 µL and the total volume in each tube is 35 µL

Add to the Vector Tube:

• 250 ng of DNA (Calculate this from the concentration of plasmid)
• 3.5 µL of appropriate 10x Buffer
• 0.5 µL of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)
• HH₂O such that the volume of water and DNA in the tube is 30.5 µL and the total volume in each tube is 35 µL

1. Mix two tubes as indicated above
2. Put the tubes into a 37°C water bath for one hour
3. Place the tubes into an 80°C heating block for 20 minutes to heat-kill the enzymes in the tube
4. Freeze the parts until they are needed

Ligation Protocol with Antarctic Phosphatase

1. Mix 5 µL of the insert and 5 &microl: of the vector in a new tube
2. Clearly label the tubes as unligated, write the date and freeze the tubes in -20°C in case the transformation does not work
3. Add 10 µL of 2x Quick Ligase Buffer and 1 µL of Quick Ligase to the tube containing the mixed Insert and Vector
4. Let the tube sit at room temperature for 5 minutes
5. Add 5 µL of 10x Antarctic Phosphatase Buffer, 4 µL of water, and 1 µL of Antarctic Phosphatase to the Vector while freezing the insert. Put the tube into a 37°C water bath for 30 minutes and then place into the 65 °C heating block for 10 minutes
6. Transform this mix (all 21 µL) into Top10 Competent Cells

Overnight Cultures
This procedure is used before the Sigma Aldrich GenElute Plasmid Preparation. You will need a 10 mL culture tube, 5 mL of LB Broth, Antibiotic, and single colonies on a plate.

1. Pipette 5 mL of LB Broth into the culture tube.
2. Add Antibiotic (10 µL Ampicillin, 5 µL Kanamycin, or 3 µL Chloramphenicol)
3. Select a single colony using a 200 µL sterile pipette tip
4. Place the culture tube into the shaker and let it shake at 175 rpm at 37°C

Glycerol Stock Preparation
This procedure is used to make long term stocks of plasmid parts that will definitely be used later on. The procedure was given by our lab technician Deirdre Lobb.

1. Grow 5 mL overnight cultures for the bacteria containing the plasmid which you wish to create glycerol stocks of (This procedure is indicated by the Overnight Growth Protocol)
2. Take 1 mL of the culture and add it to 1 mL of autoclaved 50% glycerol
3. Divide this solution into two tubes holding 1 mL each and store these in 1.5 mL microcentrifuge tubes
4. Use dry ice to flash freeze the tubes and store the glycerol stocks in a -80°C freezer

LB Agar Plate Preparation Protocol

1. Autoclave mixture of dH2O and agar
2. Add either 1 mL of Ampicillin, 0.5 mL Kanamycin or 0.35 mL Chloramphenicol
3. Pour plates, flame and mark plates
4. Let dry overnight

QIAprep Spin Miniprep Kit
This protocol is taken from the Qiagen distributed QIAprep Spin Miniprep Kit. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer. We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice. Another company's kit was used occasionally to prep the plasmids but the two kits were never mixed.

1. Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol)
2. After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4°C
3. Discard the supernatant, while being careful not to discard any of the pellet
4. Resuspend the pellet in 250 µL of Buffer P1 (with RNase A added) which is provided from the kit
5. Transfer the solution from a Falcon tube to a 1.5 µL microcentrifuge tube
6. Add 250 µL of Buffer P2 and invert gently to mix. Allow the mixture to clear for less than 5 minutes
7. Add 350 µL of Buffer N3 and invert the tube 4-6 times to mix
8. Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The resulting solution will be known as the lysate
9. Add 500 µL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube
10. Transfer the lysate into the QIAprep spin column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid
11. Centrrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube
12. Add 750 µL of Buffer PE with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube
13. Repeat Step 12 a second time with the same quantity of Wash Solution
14. Centrifuge the tube for 1 minute at 14 000 rpm to dry the column
15. Transfer the column to a new 1.5 µL microcentrifuge tube
16. Add 50 µL of double distilled water to the column and spin for 1 minute at 14 000 rpm
17. Use a spectrophotometer to measure the concentration and the purity of your plasmid

Gel Extraction
This protocol is utilized in accordance to the manufacturer's protocol from Omega E.Z.N.A (EaZy Nucleic Acid Isolation)

1. Place gel on the UV box
2. Carefully extract the fragment suspended in the gel>/li>
3. Mass gel fragments
4. Place fragment into a 1.5 mL tube and add 4 µL of H2O
5. Volume of water added to volume of gel is 200% however if fragment it small 1 mL of water will suffice
6. Remove H2O
7. Add equal amounts of H2O and Binding Buffer (XP2) to the gel
8. Incubate mixture at 55 degrees for 7 mins
9. Mix with vortex for 2 mins
10. Place in the HiBind DNA Mini Column in the 2 mL tube
11. Add 700 µL at 10,000xg for 1 min
12. Discard liquid
13. Add 300 µL Binding Buffer (XP2) into the HiBind DNA Mini Column and spin down at 10,000xg for 1 min
14. Discard liquid
15. Wash the column with 700 µL of SPW buffer with added ethanol and spin down at 10,000xg for 1 min
16. Discard liquid
17. Wash the column with 700 µL of SPW buffer again and spin down at 10,000xg for 1 min
18. Discard the liquid
19. Spin down the column at 13,000xg for 1 min to dry the column
20. Elute in 50 µL of H2O and wait 1 min
21. Spin down the column at 13,000xg for 1 min to dry the column
22. Use a spectrophotometer to measure the concentration and the purity of your plasmid

PCR Purification (Vacuum)
This protocol is utilized in accordance to the manufacturer's protocol from Qiagen PCR Vacuum Prep Kit

1. Add PCR product
2. Distribute liquid evenly by evenly tapping
3. Vacuum for 15-20 mins to dry
4. Add 20 µL H2O
5. Tap lightly against the table to redistribute the H2O on the surface of the well
6. Pipette contents and transfer to clean 1.5 mL tube